Kits and methods for assessing skin health

ABSTRACT

The invention relates to kits and methods for assessing skin health for a human and the human&#39;s susceptibility to skin disorders. The methods involve assessing occurrence in the human&#39;s genome of one or more polymorphisms (e.g., single nucleotide polymorphisms) that occur in one or more genes associated disclosed herein and that are associated with a disorder in humans. Preferred assessment and scoring methods are disclosed, as are kits for performing the methods.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation-in-part of International ApplicationNo. PCT/US02/10682 (filed 5 Apr. 2002) and of U.S. patent applicationSer. No. 09/826,522 (filed 5 Apr. 2001), and is entitled to prioritypursuant to 35 U.S.C. § 119(e) to U.S. provisional patent applicationsNo. 60/289,169 (filed 7 May 2001), No. 60/350,517 (filed 22 Oct. 2001),No. 60/335,426 (filed 24 Oct. 2001), and No. 60/336/815 (filed 5 Dec.2001).

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

Not applicable.

REFERENCE TO A MICROFICHE APPENDIX

Not applicable.

BACKGROUND OF THE INVENTION

Skin is the largest and most visible organ of the human body, and isalso among the tissues most exposed to environmental stresses, hazards,and pathogens. Skin is a multi-layered tissue, primarily composed of theepidermis and dermis, and includes several accessory structures, such assweat glands, sebaceous glands, and hair follicles. Skin serves manyfunctions. For example, it is a protective barrier to external insults(e.g., heat, chemicals, bacteria), is involved in thermoregulation,inhibits dehydration, and performs sensory functions. Skin is also abioreactor that produces various hormones and lipids that enter thebody's circulation. A variety of immune cells function in skin as afirst line of defense against bacterial or viral invasion and tomaintain immune surveillance in skin and nearby body tissues. For thesereasons, establishment and maintenance of good skin health is importantto human health.

Skin health is also important for aesthetic reasons. Many people aredeeply concerned about the appearance of their skin. A healthy skinappearance is maintained by a combination of cleaning, nutrition, andapplication of therapeutic and cosmetic products. However, overuse ofskin care products can degrade skin health and appearance. Often,individuals employ trial-and-error techniques to identify skin careproducts (and doses thereof) that produce a desirable skin appearance.More precise methods are needed for identifying compositions (andsuitable amounts of such compositions) that will enhance the health andappearance of an individual's skin. These methods would preferably betailored to identify useful compositions and dosages for individuals.The present invention satisfies this need.

Many skin disorders can be alleviated, inhibited, or even prevented bymaintaining a high degree of skin health or by timely intervention withappropriate skin-affecting agents. For example, such intervention caninclude consuming or topically applying skin care products, modulatingsun exposure, adjusting diet, consuming nutritional or pharmaceuticalproducts known to be effective against skin disorders, and undergoingheightened medical monitoring. These changes are often not made, owingto the expense or inconvenience of the changes and an individual'ssubjective belief that he or she is not at high risk for skin disorders.Improved assessment of skin health can help to identify individuals atrisk for developing skin disorders and permit more informed decisions tobe made regarding whether lifestyle changes or other interventions arejustified.

Many human genes occur in a variety of forms which differ in at leastminor ways. Heterogeneity in human genes is believed to have arisen, inpart, from minor, non-fatal mutations that have occurred in the genomeover time. In some instances, differences between alternative forms of agene are manifested as differences in the amino acid sequence of aprotein encoded by the gene. Some amino acid sequence differences canalter the reactivity or substrate specificity of the protein.Differences between alternative forms of a gene can also affect thedegree to which (if at all) the gene is expressed. However, manyheterogeneities that occur in human genes appear not to be correlatedwith any particular phenotype. Known heterogeneities include, forexample, single nucleotide polymorphisms (i.e., alternative forms of agene having a difference at a single nucleotide residue). Other knownpolymorphic forms include those in which the sequence of larger (e.g.,2-1000 residues) portions of a gene exhibits numerous sequencedifferences and those which differ by the presence or absence of portionof a gene.

Numerous disorders and physiological states have been correlated withoccurrence of one or more alternative forms of an individual gene in thegenome of a human who exhibits the disorder or physiological state. Forexample, Kimura et al. (2000, Am. J. Ophthalmol. 130:769–773) disclosesan association between occurrence of a SNP of the manganese superoxidedismutase gene and a form of macular degeneration.

Associations between individual disorders and individual geneticpolymorphisms are known. However, disorders can usually result frompolymorphisms in any of a relatively large number of genes, and as aresult, assessing the polymorphic form(s) of any single gene that occurin a human's genome is usually not predictive of the likelihood that thehuman will develop the disorder.

A need remains for a method of assessing an individual's skin health orpredisposition to develop skin disorders. Such assessment could be usedto identify types and amounts of therapeutic, inhibitory, or preventivecompositions or interventions that can be used to alleviate, inhibit, orprevent skin disorders. The invention satisfies these needs.

BRIEF SUMMARY OF THE INVENTION

The invention relates to a method of assessing skin health in a human.The method comprises assessing occurrence in the human's genome ofdisorder-associated polymorphisms in at least two (three, four, five,six, eight, ten, fifteen, or even all) genes selected from the groupconsisting of

-   -   genes which encode an enzyme that catalyzes conversion of a        toxic oxygen species to a less toxic oxygen species;    -   genes which encode a protein that provides protection against        oxidative stress;    -   genes which encode a protein that induces production of a toxic        oxygen species;    -   genes which encode a protein that indirectly affects oxidative        stress;    -   genes which encode a protein for which the level of expression        of the protein is associated with oxidative stress;    -   genes which encode a component of the human DNA repair system;        and    -   genes which encode a protein associated with production of a        toxic oxygen species by a macrophage or polymorphonuclear        neutrophilic granulocyte.

It has been discovered that this method is particularly useful forassessing skin health when the genes are selected from the groupconsisting of

-   a) the gene which encodes mitochondrial manganese superoxide    dismutase (MnSOD);-   b) the gene which encodes cytoplasmic copper/zinc superoxide    dismutase (CZSOD);-   c) the gene which encodes catalase;-   d) the gene which encodes human glutathione peroxidase (hGPX1);-   e) the gene which encodes glutathione S transferase P1 (GSTP1)-   f) the gene which encodes NAD(P)H:quinone oxidoreductase;-   g) the gene which encodes epoxide hydrolase;-   h) the gene which encodes tumor necrosis factor alpha (TNF-alpha);-   i) the gene which encodes NADH/NADPH oxidase p22 subunit (the phox    gene);-   j) the gene which encodes nitric oxide synthase;-   k) the gene which encodes cytochrome P450;-   l) the gene which encodes matrix metalloproteinase 1 (MMP-1); and-   m) the gene which encodes profilagrin.    Occurrence of a disorder-associated polymorphism in any of these    genes is an indication that the human has poorer skin health than a    human whose genome does not comprise the disorder-associated    polymorphism, and occurrence of a plurality of disorder-associated    polymorphisms is an indication that the human has even poorer skin    health than a human whose genome comprises only one of the    disorder-associated polymorphisms (and greater still than an    individual whose genome does not comprise one of these    disorder-associated polymorphisms).

Substantially the same method can be used to assess the advisabilitythat a human should employ a skin care product, such as one comprising askin protective ingredient or a vitamin (e.g., one of vitamins C and E).When the method is used to assess the advisability that a human shouldemploy a skin care product, occurrence of one or moredisorder-associated polymorphisms in any of genes a)-1) is an indicationthat it is more advisable for the human to use the product than when theindividual's genome does not comprise disorder-associated polymorphismsin any of these genes.

For example, occurrence of at least two (three, four, five, six, eight,ten, or fifteen or more) disorder-associated polymorphisms can beassessed, where the polymorphisms are selected from the group consistingof

-   A) a polymorphism in the open reading frame encoding mitochondrial    MnSOD;-   B) a polymorphism in the open reading frame encoding cytoplasmic    CZSOD;-   C) a polymorphism in the promoter region of the gene encoding    catalase;-   D) a polymorphism in the open reading frame of the hGPX1 gene;-   E) a polymorphism in the open reading frame encoding glutathione S    transferase P1 (GSTP1);-   F) a polymorphism in the open reading frame encoding NAD(P)H:quinone    oxidoreductase;-   G) a polymorphism in the open reading frame encoding epoxide    hydrolase;-   H) a polymorphism in the promoter region of the gene encoding    TNF-alpha;-   I) a polymorphism in the open reading frame of the phox gene;-   J) a polymorphism in the open reading frame encoding nitric oxide    synthase;-   K) a polymorphism in the 5′ flanking region of the gene encoding    cytochrome P450; and-   L) a polymorphism in the promoter region of the gene encoding MMP-1.

Preferably, occurrence of all known polymorphisms at individual sites(e.g., both of two known alternative forms or all three forms of apolymorphism known to exist in three alternative forms) is assessedwithin an individual's genome, so that the individual's genotype for thepolymorphism as that site can be completely known. For example,appropriate polymorphisms that can be assessed in the genes listed aboveinclude the following

-   i) a polymorphism manifested as occurrence of a codon encoding    alanine at amino acid residue 9 (i.e., in the signal sequence) of    MnSOD;-   ii) a polymorphism manifested as occurrence of a codon encoding    valine at amino acid residue 9 (i.e., in the signal sequence) of    MnSOD;-   iii) a polymorphism manifested as occurrence of a codon encoding    isoleucine at amino acid residue 58 of MnSOD;-   iv) a polymorphism manifested as occurrence of a codon encoding    thymine at amino acid residue 58 of MnSOD;-   v) a polymorphism manifested as occurrence of a codon encoding    valine at amino acid residue 7 of CZSOD;-   vi) a polymorphism manifested as occurrence of a codon encoding    glutamic acid at amino acid residue 7 of CZSOD;-   vii) a polymorphism manifested as occurrence of a codon encoding    cysteine at amino acid residue 6 of CZSOD;-   viii) a polymorphism manifested as occurrence of a codon encoding    phenylalanine at amino acid residue 6 of CZSOD;-   ix) a polymorphism manifested as occurrence of a cytosine residue at    nucleotide residue-262 (i.e., in the promoter region) of the    catalase gene;-   x) a polymorphism manifested as occurrence of a thymine residue at    nucleotide residue-262 (i.e., in the promoter region) of the    catalase gene;-   xi) a polymorphism manifested as occurrence of a codon encoding    proline at amino acid residue 198 of glutathione peroxidase (hGPX1);-   xii) a polymorphism manifested as occurrence of a codon encoding    leucine at amino acid residue 198 of glutathione peroxidase (hGPX1);-   xiii) a polymorphism manifested as occurrence of a codon encoding    valine at amino acid residue 105 of glutathione S-transferase P1    (GSTP1);-   xiv) a polymorphism manifested as occurrence of a codon encoding    isoleucine at amino acid residue 105 of glutathione S-transferase P1    (GSTP1);-   xv) a polymorphism manifested as occurrence of a cytosine residue at    nucleotide residue 242 (i.e., in the coding region) of the gene    encoding NAD(P)H:quinone oxidoreductase;-   xvi) a polymorphism manifested as occurrence of a thymine residue at    nucleotide residue 242 (i.e., in the coding region) of the gene    encoding NAD(P)H:quinone oxidoreductase;-   xvii) a polymorphism manifested as occurrence of a thymine residue    at nucleotide residue 113 in exon 3 of the gene which encodes    epoxide hydrolase (i.e., resulting in a tyrosine residue in epoxide    hydrolase);-   xviii) a polymorphism manifested as occurrence of a cytosine residue    at nucleotide residue 113 in exon 3 of the gene which encodes    epoxide hydrolase (i.e., resulting in a histidine residue in epoxide    hydrolase);-   xix) a polymorphism manifested as occurrence of an adenine residue    at nucleotide residue-238 (i.e., in the promoter region) of the gene    which encodes TNF-alpha (i.e., the TNF-alpha promoter variant    designated TNF2);-   xx) a polymorphism manifested as occurrence of an adenine residue at    nucleotide residue-308 (i.e., in the promoter region) of the gene    which encodes TNF-alpha (i.e., the TNF-alpha promoter variant    designated TNF3);-   xxi) a polymorphism manifested as occurrence of a cytosine residue    at nucleotide residue 242 (i.e., in the coding region) of the phox    gene encoding the NADH/NADPH oxidase p22 subunit;-   xxii) a polymorphism manifested as occurrence of a thymine residue    at nucleotide residue 242 (i.e., in the coding region) of the phox    gene encoding the NADH/NADPH oxidase p22 subunit;-   xxiii) a polymorphism manifested as a 27 nucleotide residue repeat    in intron 4 (i.e., between nucleotide residues 5130 and 5511) of the    gene encoding nitric oxide synthase;-   xxiv) a polymorphism manifested as absence of a 27 nucleotide    residue repeat in intron 4 (i.e., between nucleotide residues 5130    and 5511) of the gene encoding nitric oxide synthase;-   xxv) a polymorphism manifested as occurrence of an adenine residue    at nucleotide residue-290 (i.e., in the 5′-flanking region) of the    gene encoding cytochrome P450 (i.e., the polymorphism designated the    CYP3A4 cytochrome P450 variant);-   xxvi) a polymorphism manifested as occurrence of a guanine residue    at nucleotide residue-290 (i.e., in the 5′-flanking region) of the    gene encoding cytochrome P450 (i.e., the polymorphism designated the    CYP3A4 cytochrome P450 variant);-   xxvii) a polymorphism manifested as occurrence of a single guanine    residue at nucleotide residue-1607 of the human gene encoding MMP-1;    and-   xxviii) a polymorphism manifested as occurrence of a two consecutive    guanine residues at a site including nucleotide residue-1607 of the    human gene encoding MMP-1.

Occurrence of an individual disorder-associated polymorphism can beassessed by first contacting a nucleic acid derived from the human'sgenome with a first oligonucleotide that anneals with higher stringencywith the disorder-associated polymorphism than with a correspondingnon-disorder-associated polymorphism and then assessing annealing of thefirst oligonucleotide and the nucleic acid. Annealing of the firstoligonucleotide and the nucleic acid is an indication that the human'sgenome comprises the disorder-associated polymorphism.

Occurrence of an individual disorder-associated polymorphism can befurther assessed by contacting the nucleic acid with a secondoligonucleotide that anneals with higher stringency with anon-disorder-associated polymorphism than with the correspondingdisorder-associated polymorphism and assessing annealing of the secondoligonucleotide and the nucleic acid. Annealing of the secondoligonucleotide and the nucleic acid is an indication that the human'sgenome comprises the non-disorder-associated polymorphism. By assessingoccurrence of both disorder-associated and non-disorder associatedpolymorphisms in an individual's genome, one can assess whether theindividual is likely homologous for the non-disorder-associatedpolymorphism, homologous for the disorder-associated polymorphism, orheterozygous for the disorder-associated polymorphism andnon-disorder-associated polymorphisms. This information can informselection of an appropriate agent or intervention and an appropriatedose, duration, or intensity of the agent or intervention for improvingskin health or alleviating, inhibiting, or preventing a skin disorder.

A skin health score can be calculated by summing, for each of theselected genes in which a disorder-associated polymorphism occurs in thehuman's genome, the product of a constant and a correlation factor. Thecorrelation factor can be one or it can, for example, represent thefraction of humans heterozygous or homozygous for thedisorder-associated polymorphism who exhibit the corresponding disorder.The skin health score represents the relative susceptibility of thehuman to a skin disorder.

The same methods can be used to assess the likelihood that a human willdevelop a skin disorder. Occurrence of any of the disorder-associatedpolymorphisms is an indication that the human is more susceptible to theskin disorder than a human whose genome does not comprise thepolymorphism, and occurrence of a plurality of the disorder-associatedpolymorphisms is an indication that the human is even more susceptibleto the skin disorder than a human whose genome does not comprise thepolymorphisms.

These methods can also be used to select a dose of a skin protectivecomposition or other prophylactic or therapeutic composition foradministration to a human. Occurrence of any of the disorder-associatedpolymorphisms is an indication that a greater dose of the compositionshould be administered to the human. The dose of the composition can beselected based on occurrence of the polymorphisms.

The invention further relates to a kit for assessing relativesusceptibility of a human to a skin disorder. The kit comprises reagentsfor assessing occurrence in the human's genome of disorder-associatedpolymorphisms in at least two (three, four, five, six, eight, ten, orfifteen or more) of the genes disclosed herein.

In another aspect, the invention relates to a method of assessing theadvisability that a human should employ a nutritional product comprisinga skin protective agent or other prophylactic or therapeuticcomposition. The method comprises assessing occurrence in the human'sgenome of disorder-associated polymorphisms in at least two (three,four, five, six, eight, ten, fifteen, or more) of the genes disclosedherein. Occurrence of any of the disorder-associated polymorphisms is anindication that it is more advisable for the human to employ thenutritional product than a human whose genome does not comprise thepolymorphism, and occurrence of a plurality of the disorder-associatedpolymorphisms is an indication that it is even more advisable that thehuman should employ the nutritional product than a human whose genomedoes not comprise the polymorphisms.

In still another aspect, the invention relates to a method of selectinga dose of a skin protective agent for administration to a human in anutritional product. The method comprises assessing occurrence in thehuman's genome of disorder-associated polymorphisms in at least two ofthe genes disclosed herein. Occurrence of any of the polymorphisms is anindication that a greater dose of the agent should be administered tothe human in the nutritional product. The dose of the agent for thenutritional product can be selected based on occurrence of thepolymorphisms.

BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWINGS

The foregoing summary, as well as the following detailed description ofpreferred embodiments of the invention, will be better understood whenread in conjunction with the appended drawings. The invention is notlimited to the precise arrangements and instrumentalities shown.

FIGS. 1A and 1B are images which depict examples of results that can beobtained by analyzing occurrence of polymorphisms in several genes. Theresults shown in FIG. 1A are derived from a hypothetical first human,and those shown in FIG. 1B are derived from a hypothetical second human.Circles represent different polymorphisms of the gene indicated to theleft of the row of circles. Filled circles indicate the presence of thepolymorphism. Non-filled circles indicate the absence of thepolymorphism. Numbers below each circle represent a correlation factorfor the polymorphism and a disease or disorder (i.e., not necessarily askin disease or disorder).

DETAILED DESCRIPTION OF THE INVENTION

The invention relates to kits and methods for assessing skin health in ahuman by assessing occurrence in the human's genome of geneticpolymorphisms that are associated with disorders (i.e., any type ofdisorder, whether a disorder of the skin or not). To better characterizethe human's genetic content, occurrence of polymorphic forms (of thesame genes) that are not associated with a disorder can also beassessed, so that one can determine whether the human is 1) homozygousfor the disorder-associated polymorphism at a genomic site, 2)heterozygous for disorder-associated and non-disorder-associatedpolymorphisms at that site, or 3) homozygous for non-disorder-associatedpolymorphisms at that site. Assessments of genomic polymorphism contentin two or more (and preferably in three, four, five, six, eight, ten,fifteen, or more) of the genes identified herein as being significant toskin health can be combined to indicate the skin health of the human.This assessment of skin health can be used to predict the likelihoodthat the human will develop, is developing, is predisposed to develop,or has already developed a skin disorder.

Crudely simplified, the methods involve determining whether multiplepolymorphisms that have been associated (by the inventors or by others)with a human disorder (i.e., a disease or pathological state, whether ofthe skin or not) occur in the genome of the human being tested. In someembodiments, the number of polymorphisms that occur in the human'sgenome are summed to yield a value; the higher the value is, the greaterthe susceptibility of the human to skin disorders is assessed to be(i.e., the poorer the human's skin health is assessed to be). In otherembodiments, a weighting factor is assigned to each polymorphism tested,and the weighting factors of polymorphisms that occur in the human'sgenome are summed to yield a value that represents relative skin health(e.g., as assessed by susceptibility to skin disorders). The weightingfactor can represent the product of a constant assigned to the gene inwhich the corresponding polymorphism occurs and a correlation factorthat describes how informative occurrence of the polymorphism is foroccurrence of the disorder with which it is associated. The weightingfactor can also be influenced by whether the human is homozygous orheterozygous for the disorder-associated polymorphism. The inventionincludes a variety of alternative methods and kits for performing themethods, as described in greater detail herein.

Definitions

As used in this disclosure, the following terms have the meaningsassociated with them in this section.

A “polymorphism” in a gene is one of the alternative forms of a portionof the gene that are known to occur in the human population. Forexample, many genes are known to exhibit single nucleotide polymorphicforms, whereby the identity of a single nucleotide residue of the genediffers among the forms. Each of the polymorphic forms represents asingle polymorphism, as the term is used herein. Other known polymorphicforms include alternative forms in which multiple consecutive orclosely-spaced, non-consecutive nucleotide residues vary in sequence,forms which differ by the presence or absence of a single nucleotideresidue or a small number of nucleotide residues, and forms whichexhibit different mRNA splicing patterns.

A “single nucleotide polymorphism” (“SNP”) is one of the alternativeforms of a portion of a gene that vary only in the identity of a singlenucleotide residue in that portion.

A “disorder-associated” polymorphism is an alternative form of a portionof a gene, wherein occurrence of the alternative form in the genome of ahuman has been correlated with exhibition by the human of a disease or apathological state, whether the disease or pathological state affectsthe skin, another tissue, or multiple tissues.

A “non-disorder-associated” polymorphism is an alternative form of aportion of a gene for which no significant positive correlation has beenmade between occurrence of the alternative form in the genome andoccurrence of a disease or a pathological state. Non-disorder-associatedpolymorphisms are sometimes designated “neutral” polymorphisms in theart.

A disorder-associated polymorphism and a non-disorder-associatedpolymorphism “correspond” with one another if the two polymorphisms aretwo alternative forms of the same portion of the gene. By way ofexample, if the identity of residue 100 of a gene is adenine in adisorder-associated polymorphism of the gene and cytosine in anon-disorder-associated polymorphism of the gene, then the twopolymorphisms correspond with one another. It is understood that theremay be three or more corresponding polymorphisms when there are morethan two alternative forms of the same portion of the gene.

A “characteristic residue” of a polymorphism is a nucleotide residue,the identity of which is known to vary among the alternative formscorresponding to the polymorphism.

A “skin disorder” is a pathological condition characterized bydysfunction, (e.g., inflammation, necrosis, abnormal proliferation,reduced elasticity, defective renewal, irritation, or infection) of someportion of the skin.

“Skin health” is a measure of the absence of a skin disorder in anindividual human (i.e., characterized by normal skin function andappearance) and the likelihood that the individual will continue toexhibit absence of a skin disorder.

“Toxic oxygen species” include, in approximate order of decreasingreactivity, hydroxyl radicals, superoxide radicals, nitric oxide, peroxynitrite (ONOO—; the product of a reaction between nitric oxide andsuperoxide radical), and hydrogen peroxide. Ordinary diatomic oxygen isnot a toxic oxygen species, as the term is used herein.

“Oxidative damage” refers to chemical reaction of a normal cellularcomponent (e.g., DNA, a protein, or a lipid) with a toxic oxygenspecies, whereby at least one normal function of the component isinhibited or eliminated. The terms “oxidative damage” and “oxidativestress” are used interchangeably herein.

A “molecular beacon oligonucleotide” is a single-strandedoligonucleotides having a fluorescent label (e.g., rhodamine, FAM, TET,VIC, JOE, or HEX) attached to the 5′-end thereof and a fluorescencequencher (e.g., TAMRA or DABCYL) attached to the 3′-end thereof (or viceversa), as described (Kostrikis et al., 1998, Science 279:1228–1229).

Two molecular beacon oligonucleotides are “spectrally distinct” if theycan be differentially detected using spectrophotometric orspectrofluorimetric methods. Examples of characteristics that can beused to differentiate spectrally distinct oligonucleotides includeabsorption or excitation wavelength, emission wavelength, andfluorescent lifetime.

An “instructional material” is a publication, a recording, a diagram, orany other medium of expression which can be used to communicate how touse a kit described herein, numerical values for weighting thesignificance of various polymorphisms that are detectable using the kit,or both. The instructional material of the kit of the invention can, forexample, be affixed to a container which contains a kit of the inventionor be shipped together with a container which contains the kit.Alternatively, the instructional material can be shipped separately fromthe container with the intention that the instructional material and thekit be used cooperatively by the recipient.

The “stringency” with which two polynucleotides anneal means therelative likelihood that the polynucleotides will anneal in a solutionas the conditions of the solution become less favorable for annealing.Examples of stringent conditions are known in the art and can be foundin available references (e.g., Current Protocols in Molecular Biology,John Wiley & Sons, N.Y., 1989, 6.3.1–6.3.6). Aqueous and non-aqueousannealing methods are described in that reference and either can beused. In general, a first pair of polynucleotides anneal with higherstringency than a second pair if the first pair is more likely to anneal(or remain annealed) as one or more of the salt concentration,temperature, and detergent concentration are increased.

With respect to a disorder, a “correlation factor” for adisorder-associated polymorphism is the fractions of humans who areheterozygous or homozygous for the polymorphism who exhibit thedisorder. The correlation factor can, alternatively, be based solely onthose who are heterozygous, solely on those who are homozygous, or onthose who are either heterozygous or homozygous.

A “non-extendable” nucleotide residue is a nucleotide residue that iscapable of being added to a polynucleotide by a polymerase (i.e., byextension of the polynucleotide in association with a complementthereof, catalyzed by the polymerase) and that, upon addition to thepolynucleotide, renders the polynucleotide incapable of being furtherextended by the polymerase.

Description

The invention relates to kits and methods for assessing the skin healthof a human by assessing occurrence in the human's genome of geneticpolymorphisms that are associated with disorders (i.e., skin disordersor other disorders). Unlike other methods that predict susceptibility toa disorder based on occurrence of a particular polymorphism in aparticular gene, a panel of informative genes and polymorphisms isdisclosed herein. Using two or more of the genes in this panel, one canassess the susceptibility of a human to a skin disorder, even if theskin disorder has not been specifically associated with occurrence of apolymorphism in the panel.

It has been discovered an individual's skin health can be assessed bydetermining the polymorphic forms of certain genes that are present inthe individual's genome. The genes which are assessed are disclosedherein. Assessment of disorder-associated polymorphisms in two or moreof these genes (preferably three, four, five, six, eight, ten, fifteen,or more of these genes) in a human's genome is predictive of the human'sskin health. The greater the number of these genes in which occurrenceof disorder-associated polymorphisms is assessed, the greater theprecision of the methods for predicting the human's skin health islikely to be. Occurrence in the individual's genome of otherpolymorphisms (e.g., ones known to be associated with occurrence of theskin disorder of interest) can also be assessed concurrently orsequentially.

Skin disorders for which the kits and methods described herein areuseful include inflammatory disorders (e.g., contact dermatitis,uticaria, atopic dermatitis, psoriasis, lichen planus, cutaneous lupuserythematosus, pemphigus, and scleroderma, sun damage (e.g., reddeningand sun bum), infectious diseases (e.g., bacterial and viralinfections), and skin tumors (e.g., keratoses, squamous cell carcinomas,basal cell carcinomas, melanomas, and Kaposi's sarcoma).

Susceptibility of an individual to a skin disorder can be affected byoxidative stress that skin cells experience. Several of the genes havingpolymorphic forms that are informative for skin health encode proteinsthat modulate the body's response to or protection from oxidativestress. For example, genes which protect against oxidative stressinclude genes which encode an enzyme that catalyzes conversion of atoxic oxygen species to a less toxic oxygen species, genes that encode aprotein that directly provides protection against oxidative damage,genes which encode a protein that indirectly provides protection againstoxidative damage, genes which encode a component of the human DNA repairsystem, and genes (not necessarily included within the preceding groups)which are associated with inducible production of reactive oxygenspecies in immune cells following microbial infection.

Polymorphisms have been identified in some, if not all, of the numerousgenes that encode components of the human DNA repair system.Disorder-associated polymorphisms in these genes can be informative forthe skin health of an individual (e.g., for susceptibility of theindividual to a skin disorder). Examples of these genes include thosewhich encode apurinic and apyrimidinic endonucleases, enzymes thatcatalyze excision of nucleotide residues damaged by ultravioletradiation, and enzymes that catalyze site specific-recombination. Manysuch genes are known, and include those listed in Wood et al., 2001,Science 291(5507):1284–1289.

Skin comprises immune cells and acts as a first line of defense againstmicrobial invasion. Genes that induce production of reactive oxygenspecies in immune cells following microbial infection include genes(e.g., genes which encode components of the human phagocyte-specificNADPH-oxidase complex) associated with respiratory burst (sometimesdesignated oxidative burst) phenomena of macrophages andpolymorphonuclear nucleophilic granulocytes, whereby toxic oxygenspecies are produced in response to invasion of a tissue by a microbe(e.g., a protozoan, or a bacterium such as a Pseudomonas, Salmonella, orSerratia bacterium or a known pathogen such as Bacillus anthracis,Escherichia coli, or Staphylococcus aureus). Also included within thisgroup are genes which are known to be aberrant in patients afflictedwith disorders that inhibit or abolish antimicrobial activities ofmacrophages (e.g., chronic granulomatous disease). Disorder-associatedpolymorphisms in substantially any of these genes can be informative ofthe susceptibility of the individual to a skin disorder, particularly askin infection or inflammatory skin disorder. Identification ofindividuals in whom such polymorphisms occur (e.g., using the methodsdescribed herein) can be used, for example, to assess whether anindividual has an elevated risk for developing a skin disorder andwhether some disorder inhibits intervention should be undertaken.

It is not critical that the gene in which the occurrence of apolymorphism occurs is recognized as being directly or indirectlyinvolved in a skin disorder. It is sufficient that an association can bemade between either the level of expression of the gene or the sequenceof the gene product and skin health of humans.

Skin disorders include allergic reactions, such as hives and contactdermatitis. Genes that encode enzymes that catalyze reactionsresponsible for decreasing electrophilic potential of allergens (ortheir metabolites), a process designated biotransformation of allergens,can affect the skin health of a human. Members of the glutathioneS-transferase (GST) family of enzymes, such as GSTP1, participate in thebiotransformation of allergens. These enzymes also catalyzeinterconversions among reactive forms of oxygen. Occurrence of one ormore polymorphism in one of these GST genes can be used to assess skinhealth of an individual.

Another protein involved in production of toxic oxygen species bycomponents of the immune system in response to allergen exposure isTNF-alpha. Allen et al. (2000, Immunogenetics 51:201–205) described apolymorphism that occurs at nucleotide residue-308 (i.e., in thepromoter region) of the gene that encodes TNF-alpha. This polymorphismcan be one of those assessed as described herein.

Among enzymes that catalyze conversion of a toxic oxygen species to aless toxic oxygen species, four are of particular relevance, namelymitochondrial MnSOD, cytoplasmic CZSOD, catalase (CAT), and glutathioneperoxidase (GP). Polymorphisms that occur in these genes are known to beassociated with various disorders (see, e.g., Kimura et al., 2000, Am.J. Ophthalmol. 130:769–773). Occurrence of disorder-associatedpolymorphisms in at least one (and preferably two, three, or all) ofthese four genes should be assessed in the methods described herein,given the importance of these genes. Similarly, the kits describedherein preferably include reagents for detecting disorder-associatedpolymorphisms in at least one (and preferably two, three, or all) ofthese four genes. In addition, the significance of occurrence ofdisorder-associated polymorphisms in these genes can be applied byassigning a greater weighting factor to disorder-associatedpolymorphisms of these genes than to disorder-associated polymorphismsin other genes disclosed herein.

Genes in which disorder-associated polymorphisms occur that are usefulfor assessing the skin health of an individual include

-   -   genes which encode an enzyme that catalyzes conversion of a        toxic oxygen species to a less toxic oxygen species;    -   genes which encode a protein that provides protection against        oxidative stress;    -   genes which encode a protein that induces production of a toxic        oxygen species;    -   genes which encode a protein that indirectly affects oxidative        stress;    -   genes which encode a protein for which the level of expression        of the protein is associated with oxidative stress;    -   genes which encode a component of the human DNA repair system;        and    -   genes which encode a protein associated with production of a        toxic oxygen species by a macrophage or polymorphonuclear        neutrophilic granulocyte.

It has been discovered that the following genes are of particularrelevance to skin health:

-   a) the gene which encodes mitochondrial MnSOD;-   b) the gene which encodes cytoplasmic CZSOD;-   c) the gene which encodes catalase;-   d) the gene which encodes hGPX1;-   e) the gene which encodes GSTP1-   f) the gene which encodes NAD(P)H:quinone oxidoreductase;-   g) the gene which encodes epoxide hydrolase;-   h) the gene which encodes TNF-alpha;-   i) the phox gene;-   j) the gene which encodes nitric oxide synthase;-   k) the gene which encodes cytochrome P450;-   l) the gene which encodes MMP-1; and-   m) the gene which encodes profilagrin.

Occurrence in a patient's genome of a disorder-associated polymorphismin one of genes a)-1) is an indication that the patient is at a greaterrisk of developing a skin disorder (or is already afflicted with thedisorder) than a human whose genome does not include thedisorder-associated polymorphism. Occurrence of multipledisorder-associated polymorphisms in these genes in a patient's genomeis an indication that that patient is at greater risk for developing askin disorder (i.e., has poorer skin health) than a human in whosegenome fewer (or none) of the disorder-associated polymorphisms occur.Thus, there is a cumulative effect of disorder-associated polymorphismsin the genes identified herein on the skin health of the human in whichthey occur.

Occurrence of two copies of the same disorder-associated polymorphism inthe same human (i.e., homozygosity for the disorder-associatedpolymorphism) is an indication that the human is at a greater risk fordeveloping a skin disorder (i.e., has poorer skin health) than a humanin whom only a single copy of the polymorphism occurs (i.e., anindividual heterozygous for the disorder-associated polymorphism).Homozygosity for the disorder-associated polymorphism can be accountedfor by more heavily weighting occurrence of two copies of thedisorder-associated polymorphism than occurrence of only a single copy(e.g., by multiplying the significance associated with occurrence of thedisorder-associated polymorphism by a factor such as two, five, ten oranother value).

Although the invention is not limited to the particulardisorder-associated polymorphisms in the genes identified herein, it isrecognized that disorder-associated polymorphisms that occur inparticular portions of the genes can be more significant indicators ofskin health than disorder-associated polymorphisms that occur inparticular portions of the genes. Thus, disorder-associatedpolymorphisms that occur in the following regions of the indicated genescan be weighted more heavily than disorder-associated polymorphisms thatoccur in other portions of the genes. These polymorphisms include

-   A) disorder-associated polymorphisms in the open reading frame    encoding mitochondrial MnSOD;-   B) disorder-associated polymorphisms in the open reading frame    encoding cytoplasmic CZSOD;-   C) disorder-associated polymorphisms in the promoter region of the    gene encoding catalase;-   D) disorder-associated polymorphisms in the open reading frame    encoding hGPX1;-   E) disorder-associated polymorphisms in the open reading frame    encoding GSTP1;-   F) disorder-associated polymorphisms in the open reading frame    encoding NAD(P)H:quinone oxidoreductase;-   G) disorder-associated polymorphisms in the open reading frame    encoding epoxide hydrolase;-   H) disorder-associated polymorphisms in the promoter region of the    gene encoding TNF-alpha;-   I) disorder-associated polymorphisms in the open reading frame of    the phox gene;-   J) disorder-associated polymorphisms in the open reading frame    encoding nitric oxide synthase;-   K) disorder-associated polymorphisms in the 5′ flanking region of    the gene encoding cytochrome P450; and-   L) disorder-associated polymorphisms in the promoter region of the    gene encoding MMP-1.

Occurrence of any of a number of particular polymorphisms can be assayedin order to assess an individual's skin health. A non-limiting list ofsuch polymorphisms include the following:

-   I) a polymorphism manifested as a change from an alanine residue to    a valine residue at amino acid residue 9 (i.e., in the signal    sequence) of mitochondrial MnSOD;-   II) a polymorphism manifested as a change from an isoleucine residue    to a thymine residue at amino acid residue 58 of mitochondrial    MnSOD;-   III) a polymorphism manifested as a change from a valine residue to    a glutamic acid residue at amino acid residue 7 of cytoplasmic    CZSOD;-   IV) a polymorphism manifested as a change from a cysteine residue to    a phenylalanine residue at amino acid residue 6 of cytoplasmic    CZSOD;-   V) a polymorphism manifested as a change from a cytosine residue to    a thymine residue at nucleotide residue-262 (i.e., in the promoter    region) of the catalase gene;-   VI) a polymorphism in the hGPX1 gene manifested as a change from a    proline residue to a leucine residue at amino acid residue 198 of    glutathione peroxidase;-   VII) a polymorphism in the GSTP1 gene manifested as a change from a    valine residue to an isoleucine residue at amino acid residue 105 of    glutathione S-transferase P1;-   VIII) a polymorphism manifested as a change from a cytosine residue    to a thymine residue at nucleotide residue 242 (i.e., in the coding    region) of the gene encoding NAD(P)H:quinone oxidoreductase;-   IX) a polymorphism manifested as a change from a thymine residue to    a cytosine residue at nucleotide residue 113 in exon 3 of the gene    which encodes epoxide hydrolase (i.e., effecting change of from a    tyrosine residue to a histidine residue in epoxide hydrolase);-   X) a polymorphism manifested as a change to an adenine residue at    nucleotide residue-238 (i.e., in the promoter region) of the gene    which encodes TNF-alpha (i.e., the TNF-alpha promoter variant    designated TNF2);-   XI) a polymorphism manifested as a change to an adenine residue at    nucleotide residue-308 (i.e., in the promoter region) of the gene    which encodes TNF-alpha (i.e., the TNF-alpha promoter variant    designated TNF3);-   XII) a polymorphism manifested as a change from a cytosine residue    to a thymine residue at nucleotide residue 242 (i.e., in the coding    region) of the phox gene encoding the NADH/NADPH oxidase p22    subunit;-   XIII) a polymorphism manifested as a 27 nucleotide residue repeat in    intron 4 (i.e., between nucleotide residues 5130 and 5511) of the    gene encoding nitric oxide synthase;-   XIV) a polymorphism manifested as a change from an adenine residue    to a guanine residue at nucleotide residue-290 (i.e., in the    5′-flanking region) of the gene encoding cytochrome P450 (i.e., the    polymorphism designated the CYP3A4 cytochrome P450 variant); and-   XV) a polymorphism manifested as occurrence of a two consecutive    guanine residues at a site including nucleotide residue-1607 of the    human gene encoding MMP-1.

Occurrence of a disorder-associated polymorphism in an individual'sgenome can be assessed in any of a variety of ways. In one embodiment, asimple test (e.g., primer extension, PCR amplification, or molecularbeacon oligonucleotide-binding) is used to determine whether or not thedisorder-associated polymorphism occurs in the individual's genome(i.e., without regard to copy number). In another embodiment, a test isused to determine whether the individual's genome includes anon-disorder-associated polymorphism corresponding to a knowndisorder-associated polymorphism in a gene disclosed herein (i.e., as anindication that the individual is at least heterozygous for thenon-disorder-associated polymorphism). In yet another embodiment, a test(i.e., using multiple probes or primers) is used that is able to detectboth disorder-associated polymorphisms and non-disorder-associatedpolymorphisms in two, three, four, or more genes disclosed herein. Usingsuch a test, one can determine both occurrence of a disorder-associatedpolymorphism in an individual's genome and whether the individual ishomozygous or heterozygous for the disorder-associated polymorphism.This test also permits ‘checking’ of results, since it can both accountfor all known polymorphic forms and indicate when a previouslyuncharacterized polymorphism occurs at or near the site of a knownpolymorphism.

In a kit or method for detecting both disorder-associated polymorphismsand non-disorder-associated polymorphisms known for the genes disclosedherein, one or more (preferably at least two, three, four, five, six,eight, ten, or fifteen or more) of the following polymorphisms can beassessed:

-   i) a polymorphism manifested as occurrence of a codon encoding    alanine at amino acid residue 9 (i.e., in the signal sequence) of    mitochondrial MnSOD;-   ii) a polymorphism manifested as occurrence of a codon encoding    valine at amino acid residue 9 (i.e., in the signal sequence) of    mitochondrial MnSOD;-   iii) a polymorphism manifested as occurrence of a codon encoding    isoleucine at amino acid residue 58 of mitochondrial MnSOD;-   iv) a polymorphism manifested as occurrence of a codon encoding    thymine at amino acid residue 58 of mitochondrial MnSOD;-   v) a polymorphism manifested as occurrence of a codon encoding    valine at amino acid residue 7 of cytoplasmic CZSOD;-   vi) a polymorphism manifested as occurrence of a codon encoding    glutamic acid at amino acid residue 7 of cytoplasmic CZSOD;-   vii) a polymorphism manifested as occurrence of a codon encoding    cysteine at amino acid residue 6 of cytoplasmic CZSOD;-   viii) a polymorphism manifested as occurrence of a codon encoding    phenylalanine at amino acid residue 6 of cytoplasmic CZSOD;-   ix) a polymorphism manifested as occurrence of a cytosine residue at    nucleotide residue-262 (i.e., in the promoter region) of the    catalase gene;-   x) a polymorphism manifested as occurrence of a thymine residue at    nucleotide residue-262 (i.e., in the promoter region) of the    catalase gene;-   xi) a polymorphism in the hGPX1 gene manifested as occurrence of a    codon encoding proline at amino acid residue 198 of glutathione    peroxidase;-   xii) a polymorphism in the hGPX1 gene manifested as occurrence of a    codon encoding leucine at amino acid residue 198 of glutathione    peroxidase;-   xiii) a polymorphism in the GSTP1 gene manifested as occurrence of a    codon encoding valine at amino acid residue 105 of glutathione    S-transferase P1;-   xiv) a polymorphism in the GSTP1 gene manifested as occurrence of a    codon encoding isoleucine at amino acid residue 105 of glutathione    S-transferase P1;-   xv) a polymorphism manifested as occurrence of a cytosine residue at    nucleotide residue 242 (i.e., in the coding region) of the gene    encoding NAD(P)H:quinone oxidoreductase;-   xvi) a polymorphism manifested as occurrence of a thymine residue at    nucleotide residue 242 (i.e., in the coding region) of the gene    encoding NAD(P)H:quinone oxidoreductase;-   xvii) a polymorphism manifested as occurrence of a thymine residue    at nucleotide residue 113 in exon 3 of the gene which encodes    epoxide hydrolase (i.e., resulting in a tyrosine residue in epoxide    hydrolase);-   xviii) a polymorphism manifested as occurrence of a cytosine residue    at nucleotide residue 113 in exon 3 of the gene which encodes    epoxide hydrolase (i.e., resulting in a histidine residue in epoxide    hydrolase);-   xix) a polymorphism manifested as occurrence of an adenine residue    at nucleotide residue-238 (i.e., in the promoter region) of the gene    which encodes TNF-alpha (i.e., the TNF-alpha promoter variant    designated TNF2);-   xx) a polymorphism manifested as occurrence of an adenine residue at    nucleotide residue-308 (i.e., in the promoter region) of the gene    which encodes TNF-alpha (i.e., the TNF-alpha promoter variant    designated TNF3);-   xxi) a polymorphism manifested as occurrence of a cytosine residue    at nucleotide residue 242 (i.e., in the coding region) of the phox    gene encoding the NADH/NADPH oxidase p22 subunit;-   xxii) a polymorphism manifested as occurrence of a thymine residue    at nucleotide residue 242 (i.e., in the coding region) of the phox    gene encoding the NADH/NADPH oxidase p22 subunit;-   xxiii) a polymorphism manifested as a 27 nucleotide residue repeat    in intron 4 (i.e., between nucleotide residues 5130 and 5511) of the    gene encoding nitric oxide synthase;-   xxiv) a polymorphism manifested as absence of a 27 nucleotide    residue repeat in intron 4 (i.e., between nucleotide residues 5130    and 5511) of the gene encoding nitric oxide synthase;-   xxv) a polymorphism manifested as occurrence of an adenine residue    at nucleotide residue-290 (i.e., in the 5′-flanking region) of the    gene encoding cytochrome P450 (i.e., the polymorphism designated the    CYP3A4 cytochrome P450 variant);-   xxvi) a polymorphism manifested as occurrence of a guanine residue    at nucleotide residue-290 (i.e., in the 5′-flanking region) of the    gene encoding cytochrome P450 (i.e., the polymorphism designated the    CYP3A4 cytochrome P450 variant);-   xxvii) a polymorphism manifested as occurrence of a single guanine    residue at nucleotide residue-1607 of the human gene encoding MMP-1;    and-   xxviii) a polymorphism manifested as occurrence of a two consecutive    guanine residues at a site including nucleotide residue-1607 of the    human gene encoding MMP-1.

Another important set of polymorphisms that can be assessed in order todetermine an overall skin health score for a human aredisorder-associated polymorphisms that occur in the human profilagringene. Numerous polymorphic forms of these gene are known, and theassociations of each of these forms with one or more disorders is notyet fully characterized. Of course, whenever a profilagrin polymorphicform is or becomes associated with a disorder, occurrenc of thatdisorder-associated polymorphic form of the profilagrin gene can be usedto assess skin health in a human. Known profilagrin polymorphismsinclude SNPs and filagrin-polymer-length polymorphisms. This latter termrefers to the number of filagrin polypeptides into which the profilagrinpolypeptide is post-translationally cleaved in a human cell. Variousindividual humans are known to harbor profilagrin genes that encode aprofilagrin with at least 9 to 12 filagrin units.

An important aspect of this invention is that human skin health (e.g.,susceptibility to a skin disorder such as psoriasis, eczema, a skincancer, or a bacterial infection) can be associated with occurrence inthe human's genome of a disorder-associated polymorphism in one of thegenes described herein—even if there is no known biochemical orphysiological association between occurrence of the polymorphism andskin health or incidence of a skin disorder. Put another way, thepresent inventors have discovered that genes and polymorphisms disclosedherein are predictive indicators of the state of an individual human'sskin health. By assessing whether or not disorder-associatedpolymorphisms occur in the genes identified herein in an individual (andhow many such polymorphisms occur in those genes), one can assess theindividual's skin health (e.g., as manifested as the likelihood that theindividual has, or will develop a skin disorder).

If it is determined that an individual has poor skin health (e.g.,because multiple disorder-associated polymorphisms occur in theindividual's genome in the genes disclosed herein), then the individualcan be encouraged to make changes to improve their skin health, skinappearance, or to reduce the likelihood of developing skin disorders.Such changes can include use of skin protective compositions (e.g.,nutritional formulas including anti-oxidants, sunscreens, and topical orsystem corticosteroids), use of cosmetic compositions, improvingnutrition, and avoiding sunlight. Determination that an individual hasrelatively poor skin health can also be used as an indication that theindividual should be monitored more closely than others for developmentof skin disorders.

Early detection of a predisposition to develop a skin disorder canenable an individual (or the individual's physician) to take steps todelay, inhibit, alleviate (i.e., reduce the severity of), or evenprevent the disorder. The appropriate steps for treating and preventingskin disorders are well known and include modifying diet, exercise, andintake or topical application of nutrients and pharmaceuticals.Palliative, therapeutic, and prophylactic methods are known for manyskin disorders, and these can be undertaken once a patient'ssusceptibility to the disorder is known. Thus, the kits and methodsdescribed herein permit a skin disorder to be treated, inhibited, orprevented. The kits and methods described herein allow theseinterventions to be made at an early stage of the skin disorder (whentreatment is often most effective), or even before the disorder issymptomatically manifested.

It was not previously appreciated that detection in a human's genome oftwo or more disorder-associated polymorphisms in the genes disclosedherein is indicative that the human exhibits poorer skin health,manifested as greater susceptibility to skin disorders than individualshaving a genome containing fewer (or none) of these disorder-associatedpolymorphisms. Previous studies are believed to have recognized onlyassociation between a polymorphism in only individual genes identifiedherein and a particular disorder. The inventors believe that they arethe first to describe methods and kits for assessing a human'ssusceptibility to skin disorders based on occurrence in the human ofcertain polymorphisms that are not recognized as being associated withthe individual skin disorder.

It has been discovered that disorder-associated polymorphisms that occurin the genes identified herein as a)-1) can be used to assess both anindividual's skin health and the likelihood that the individual willdevelop (or is currently afflicted with) a skin disorder. In oneembodiment of the kits and method described herein, occurrence ofdisorder-associated polymorphisms (and/or non-disorder-associatedpolymorphisms) is assessed in two or more of the genes identified hereinas a)-1), such as occurrence of a disorder-associated polymorphismsidentified herein as A)-1). By way of example, the kit or method caninvolve assessing occurrence of multiple polymorphisms identified hereinas i)-xxviii).

Methods of Assessing Skin Health

The invention includes a method of assessing the skin health (e.g.,relative susceptibility to one or more skin disorders) of a human. Skinhealth can be calculated relative to a hypothetical human whose genomedoes not contain a single disorder-associated polymorphism in a genedisclosed herein. Alternatively, susceptibility can be calculatedrelative to another human who may have one or more differentdisorder-associated polymorphism than the human being assessed. Inpractice, the basis upon which raw susceptibility scores are calculatedis immaterial, so long as the same basis is used for all humans whosescores are to be compared (i.e., so that the scores are relatable to oneanother).

The relative skin health of a human can be used to assess the risks andbenefits of a variety of compositions, conditions, and interventions. Inone embodiment, the skin health of a human can be used to determinewhether the human would benefit by supplementing nutritional intake witha composition that contains one or more vitamins, minerals, or otherskin protective agents. Numerous skin protective agents are known andadditional agents are certain to be discovered over time. The usefulnessof the kits and methods disclosed herein does not depend on the identityof the particular agent. Examples of skin protective agents includevitamins (especially anti-oxidant vitamins), minerals,naturally-occurring amino acids, derivatives of naturally-occurringamino acids, plant extracts, and conventional skin care products (e.g.,skin softening and moisturizing lotions, Aloe extracts, and the like).Anti-oxidant vitamins are preferably administered to skin in aprotein-complexed form (e.g., using preparations such as the VITAZYME®vitamin preparations sold by Arch Personal Care Products, L.P. of SouthPlainfield, N.J.). Similarly, skin protective minerals such as manganeseand selenium are also preferably administered to skin in aprotein-complexed form (e.g., using preparations such as theACQUA-BIOMIN™ mineral preparations sold by Arch Personal Care Products,L.P.). Useful skin protective plant extracts include gape polyphenolsand naturally active botanicals (NABs) such as NAB Pikea robusta (redalgae) extract, NAB fennel seed (Foeniculum vulgare) extract, and NABred clover (Trifollum Pratense) leaf extract. Useful naturally-occurringamino acids and derivatives thereof include glycine, glutamine,N-acetylcysteine, and trimethylglycine. Furthermore, the skin health, asassessed using a kit or method as described herein, can indicate anappropriate dose of such an agent for an individual patient.

The skin protective agent that is administered to an individual subjectcan be determined by the overall skin health score, by observing thegenes in which disorder-associated polymorphisms occur, or both.

For example, if a disorder-associated polymorphism occurs in thesubject's MnSOD gene, then a manganese-containing skin protective agent,a zinc-containing skin protective agent, or a manganese- andzinc-containing skin protective agent (e.g., one of the ACQUA BIOMIN™products) can be applied to the subject's skin to inhibit or alleviateskin disorders.

If a disorder-associated polymorphism occurs in the subject'sglutathione peroxidase gene, then a skin protective agent comprising oneor more of selenium, grape polyphenols, N-acetylcysteine, glutamine,glycine, or NAB fennel seed can be applied to the subject's skin toinhibit or alleviate skin disorders.

If a disorder-associated polymorphism occurs in the subject's microsomalepoxide hydrolase gene, then a skin protective agent comprising one ormore of N-acetylcysteine, trimethylglycine, an anti-oxidant vitamine(e.g., one of the VITAZYME® products), NAB Pikea robusta, and NAB fennelseed can be applied to the subject's skin to inhibit or alleviate skindisorders.

If a disorder-associated polymorphism occurs in the subject's tumornecrosis factor-alpha gene, then a skin protective agent comprising oneor both NAB Pikea robusta and NAB red clover leaf can be applied to thesubject's skin to inhibit or alleviate skin disorders.

Skin health of a human is determined by assessing occurrence in thehuman's genome of disorder-associated polymorphisms in a plurality ofgenes disclosed herein (e.g., 2, 3, 4, 6, 8, 10, 15, or more genes).Occurrence of a disorder-associated polymorphism in one of these genesis an indication that the human has a greater susceptibility to skindisorders and poorer skin health than a human in whose genome thepolymorphism does not occur. Occurrence of two or more suchpolymorphisms in the human's genome indicates that the human exhibitseven greater susceptibility to skin disorders (and poorer skin health).

Occurrence of each disorder-associated polymorphism in a gene disclosedherein is not necessarily equally indicative of susceptibility to skindisorders and poorer skin health. In order to account for differences inthe significance of various disorder-associated polymorphisms, aweighting factor can be assigned to each polymorphism detected in themethods and kits described herein. As indicated above, some genes have amore significant role in skin health in humans than others. Generally,disorder-associated polymorphisms that occur in one of these genes aremore significant than polymorphisms that occur in genes having lesssignificant roles in skin health. Thus, a greater weighting factor canbe assigned to these polymorphisms than to others. By way of example,the weighting factor assigned to these polymorphisms can be 1 to 10times greater than the weighting factor assigned to disorder-associatedpolymorphisms in other genes.

Another factor which can influence the significance that is assigned tooccurrence of a disorder-associated polymorphism in a human's genome isthe degree to which the polymorphism is correlated with thecorresponding disorder. Some disorders are highly correlated withoccurrence of a genetic polymorphism, and other disorders exhibit lowercorrelation with a polymorphism. When a polymorphism is reported to beassociated with a disorder (i.e., with a disease or pathologicalcondition), a degree of correlation between the polymorphism and thedisorder can be determined or obtained from reports in the literature.One useful way of calculating a factor that describes correlationbetween a polymorphism and a disorder is to calculate an odds ratio thatdescribes the likelihood that an individual in whose genome thedisorder-associate polymorphism occurs will exhibit or develop thedisorder. Because the kits and methods described herein can be used todetect whether the human is homozygous for the disease-associatedpolymorphism, odds ratios calculated for homozygous individuals can alsobe used, if they are available. Odds ratios can be calculated asdescribed in the art.

For a disorder-associated polymorphism, the odds ratio can be calculatedas follows. First, the odds of being afflicted with the disorder arecalculated for a first population in which the polymorphism occurs bydividing the number of afflicted individuals in the first population bythe total number of individuals in the first population. Second, theodds of being afflicted with the disorder are calculated for a firstpopulation in whom the polymorphism does not occur by dividing thenumber of afflicted individuals in the second population by the totalnumber of individuals in the second population. Third, the odds ratio iscalculated by dividing the odds for the first population by the odds forthe second population. If the odds ratio is greater than one, then thisis an indication that occurrence of the polymorphism is associated withoccurrence of the disorder. Furthermore, the magnitude of the odds ratiois an indication of the significance of the association.

A skin health score for a human can be determined as follows. Asignificance score can be assigned to each disorder-associatedpolymorphism that is detected in the human's genome using a method orkit described herein. The significance score is a constant (e.g., 1.00),and is multiplied by any significance factor (e.g., 1–10) and by anycorrelation factor that is available. If information is available whichdescribes the correlation between homozygosity for the polymorphism andthe corresponding disorder, then that correlation factor should be usedin place of the correlation factor for mere occurrence of thepolymorphism, at least if the method or kit is used to rule outoccurrence in the subject's genome of correspondingdisorder-non-associated polymorphisms. If significance and correlationfactors are not available, then values of 1.00 should be assigned toeach. The skin health score is determined by summing the significancescore for each disorder-associated polymorphism that is detected usingthe method or kit. This skin health score can be compared with thevalues obtained from other subjects, or it can be compared with thevalue (i.e., zero) which would be expected to occur in a human whosegenome does not include any disorder-associated polymorphism in a genedisclosed herein. A high skin health score corresponds to poor skinhealth. Thus, for two individuals having different skin health scores,the individual having the lower score has better skin health than theindividual having the higher score.

The method used to assess occurrence of any particulardisorder-associated polymorphism (or disorder-non-associatedpolymorphism) is not critical. Numerous methods of detecting occurrenceof a polymorphism are known in the art, and substantially any of thosemethods can be used in the kits and methods described herein. Naturally,the reagents included in the kit will vary depending on the method to beused to detect the polymorphisms. Examples of some suitable polymorphismdetection methods are provided below.

In one embodiment, a pair of oligonucleotide primers are used to amplifya portion of the gene that includes a polymorphic region. Detection ofone or more of the polymorphisms that occur at the polymorphic regioncan be achieved by contacting the amplified portion with anoligonucleotide having a sequence such that it will anneal understringent conditions with the amplified portion only if one polymorphismoccurs at the portion, but will not anneal with the amplified portion ifanother polymorphism occurs at that portion. Various acceptablestringent conditions are known in the art, and can be modified by theskilled artisan as appropriate to any particular amplifiedportion/oligonucleotide pair. An example of stringent conditions ishybridization in 6× sodium chloride/sodium citrate (SSC) at about 45°C., followed by one or more washes in 0.2×SSC, 0.1% (w/v) SDS at 50° C.

In an alternative embodiment, one or more molecular beaconoligonucleotides are used to detect polymorphisms (disorder-associated,non-disorder-associated, or both) in a sample that contains a copy ofthe subject's genome, a fraction of the subject's genome, oramplification products generated from the subject's genome (e.g.,amplified portions of one or more of the genes disclosed herein in whichpolymorphisms are known to occur).

Molecular beacon probes are single-stranded oligonucleotides having afluorescent label (e.g. rhodamine, FAM, TET, VIC, JOE, or HEX) attachedat or near the 5′-end thereof and a fluorescence quencher (e.g. TAMRA orDABCYL) attached at or near the 3′-end thereof (or vice versa), asdescribed (Kostrikis et al., 1998, Science 279:1228–1229). The sequenceof each molecular beacon probe is selected to include two complementaryhairpin regions, whereby the probe can self-anneal to form a hairpinstructure. The 5′-and 3′-ends are brought into close association whenthe hairpin structure forms. The probe also comprises a targetingportion which is selected to be complementary to a target sequence (e.g.a single polymorphism of a gene disclosed herein). The targeting portionand at least one of the hairpin regions are located in close proximityto one another, meaning that the targeting portion either overlaps thehairpin region or flanks it, having no more than about 5 nucleotideresidues therebetween.

If the hairpin regions of the molecular beacon probe anneal with oneanother, then the probe does not fluoresce, because the hairpinstructure forms and the fluorescence quencher attached to one end of theprobe quenches fluorescence of the label attached to the other end ofthe probe. If the targeting portion of the probe anneals with a regionof a nucleic acid having the target sequence, then formation of thehairpin structure is inhibited, the fluorescence quencher is not broughtinto association with the fluorescent label, and the probe fluoresces.Multiple molecular beacon probes can be used in a single reactionmixture, and fluorescence associated with the probes can bedifferentiated if the molecular beacon probes are spectrally distinct.

Thus, in this embodiment, one or more molecular beacon probes are used,each having targeting portion which is complementary to a target region(e.g. 20 to 40 nucleotide residues, more preferably 20 to 30 residues)of one polymorphism of a gene disclosed herein. If the polymorphism tobe detected is a single nucleotide polymorphism (SNP), then the targetregion includes, and preferably is approximately centered around, thenucleotide residue at which the polymorphism occurs. More preferably,two such probes are used, one having a targeting region completelycomplementary to the target region of one polymorphism of the gene(e.g., one of two polymorphisms of a particular SNP), and the otherhaving a targeting region completely complementary to the target regionof a corresponding polymorphism of the gene (e.g., the otherpolymorphism of the SNP if there are only two polymorphic forms), sothat occurrence of disorder-associated and non-disorder-associatedpolymorphisms can be simultaneously determined.

In yet another embodiment of how polymorphisms in gene disclosed hereincan be assessed, oligonucleotide primers which are complementary to aregion adjacent a characteristic residue of the polymorphism areextended using a polymerase enzyme, and the identity of the nucleotideresidue that is added to the primer in the position complementary to thecharacteristic residue is determined. The primer can be extended in thepresence of non-extendable nucleotide residues in order to ensure that alimited number of nucleotide residues (or only one) are incorporatedinto the primer. Methods of this type are known in the art (e.g., theSNP-IT® technology of Orchid Biocomputer, Inc.) and are described, forexample in U.S. Pat. Nos. 6,013,431 and 6,004,744.

Methods of Assessing Susceptibility to Individual Skin Disorders

An patient's skin health score is predictive of the patient'ssusceptibility to individual skin disorders (a higher score indicating agreater susceptibility to such disorders). The rate or likelihood ofdevelopment and progression of skin disorders can be estimated byassessing the skin health (i.e., determining a skin health score) of apatient. The rate or likelihood of development and progression of theparticular skin disorders disclosed herein can be estimated by assessingoccurrence of the disorder-associated polymorphisms disclosed herein.

The individual skin disorders for which susceptibility can be assessedusing these methods are not limited to those disclosed herein. Themethods can be used to assess susceptibility to substantially any skindisorder. However, it is likely that congenital skin defects which leadto development of aberrant skin in utero or during the first few yearsof life are unlikely to be associated with the disorder-associatedpolymorphisms described herein.

Kits for Assessing Skin Health

The invention includes a kit for assessing the skin health of a humanand/or the susceptibility of the human to a skin disorder. The kitcontains reagents for performing one or more of the methods describedherein. The reagents used in certain embodiments of the methodsdescribed herein are indicated above. Reagents useful for performingthose methods using a variety of alternative sample preparation andpolymorphism detection methods or chemistries are apparent to theskilled artisan.

Kits for detecting polymorphisms in individual genes are known in theart, and the kit of the invention can have similar components. However,a critical feature of the kit is that it includes reagents that permitits user to detect disorder-associated polymorphisms in at least two (orat least three, four, six, eight, ten, or fifteen or more) genesdisclosed herein.

In one embodiment, the kit includes a plurality of oligonucleotideswhich anneal under stringent conditions with a disorder-associatedpolymorphism of one of the genes (e.g., one of the genes identifiedherein as being of particular relevance for skin health), but not with anon-disorder associated-polymorphism. Each of the oligonucleotides canbe attached to a surface in order to facilitate handling of theoligonucleotide. The oligonucleotides can be linked with a plurality ofsurfaces (e.g., oligonucleotides for a particular polymorphism beingattached to a particle discrete from a particle to whicholigonucleotides for another polymorphism are attached), or they can beattached to discrete regions of a single surface (e.g., a glass orsilicon surface having oligonucleotides attached at defined locationsthereon, as in the GENECHIP™ device of Affymetrix, Inc.). Annealingbetween individual oligonucleotides and the polymorphism correspondingthereto can be detected using standard methods. The kit can alsocomprise oligonucleotides that are useful as molecular beacon probes oras extendable primers.

In one embodiment, the kit further comprises a DNA collection kit orapparatus, such as that described in co-pending U.S. Pat. No. 6,291,171.Advantageously, DNA collected using the kit or apparatus can be storedor archived, and subjected to additional testing as previously unknowndisorder-associated polymorphisms are discovered in the genes disclosedherein, or as the significance of previously unappreciated polymorphismsis realized.

The invention also relates to a method of assessing the advisabilitythat a human should consume or apply a nutritional product comprising askin protective agent such as those described above. The method isperformed as described herein for assessing the skin health of a human.If poorer skin health is detected in the human (i.e., relative to ahuman not having a disorder-associated polymorphism in a gene identifiedherein), then it is more advisable the human should consume or apply anutritional product comprising the skin protective agent. A greater skinhealth score (i.e., corresponding to poorer skin health) in a humancorrelates with an increased advisability that the human should use sucha nutritional product, and also indicates that a greater dose of theskin agent(s) should be included in the nutritional product.

It will be appreciated by those skilled in the art that changes can madeto the embodiments described above without departing from the broadinventive concept thereof.

The disclosure of every patent, patent application, and publicationcited herein is hereby incorporated herein by reference in its entirety.

This invention is not limited to the particular embodiments disclosed,and includes modifications within the spirit and scope of the presentinvention as defined by the appended claims.

1. A method of assessing the advisabilitv that a human should apply aproduct comprising a skin protective agent, the method comprisingassessing occurrence in the human's genome of at least threepolymorphisms selected from the group consisting of i) a polymorphismmanifested as occurrence of a codon encoding alanine at amino acidresidue 9 of mitochondrial MnSOD; ii) a polymorphism manifested asoccurrence of a codon encoding valine at amino acid residue 9 ofmitochondrial MnSOD; iii) a polymorphism manifested as occurrence of acodon encoding isoleucine at amino acid residue 58 of mitochondrialMnSOD; iv) a polymorphism manifested as occurrence of a codon encodingthymine at amino acid residue 58 of mitochondrial MnSOD; v) apolymorphism manifested as occurrence of a codon encoding valine atamino acid residue 7 of cytoplasmic CZSOD; vi) a polymorphism manifestedas occurrence of a codon encoding glutamic acid at amino acid residue 7of cytoplasmic CZSOD; vii) a polymorphism manifested as occurrence of acodon encoding cysteine at amino acid residue 6 of cytoplasmic CZSOD;viii) a polymorphism manifested as occurrence of a codon encodingphenylalanine at amino acid residue 6 of cytoplasmic CZSOD; ix) apolymorphism manifested as occurrence of a cytosine residue atnucleotide residue-262 of the catalase gene; x) a polymorphismmanifested as occurrence of a thymine residue at nucleotide residue-262of the catalase gene; xi) a polymorphism manifested as occurrence of acodon encoding proline at amino acid residue 198 of hGPX1; xii) apolymorphism manifested as occurrence of a codon encoding leucine atamino acid residue 198 of hGPX1; xiii) a polymorphism manifested asoccurrence of a codon encoding valine at amino acid residue 105 ofGSTP1; xiv) a polymorphism manifested as occurrence of a codon encodingisoleucine at amino acid residue 105 of GSTP1; xv) a polymorphismmanifested as occurrence of a cytosine residue at nucleotide residue 242of the gene encoding NAD(P)H:quinone oxidoreductase; xvi) a polymorphismmanifested as occurrence of a thymine residue at nucleotide residue 242of the gene encoding NAD(P)H:quinone oxidoreductase; xvii) apolymorphism manifested as occurrence of a thymine residue at nucleotideresidue 113 in exon 3 of the gene which encodes epoxide hydrolase;xviii) a polymorphism manifested as occurrence of a cytosine residue atnucleotide residue 113 in exon 3 of the gene which encodes epoxidehydrolase; xix) a polymorphism manifested as occurrence of an adenineresidue at nucleotide residue -238 of the gene which encodes TNF-alpha;xx) a polymorphism manifested as occurrence of an adenine residue atnucleotide residue -308 of the gene which encodes TNF-alpha; xxi) apolymorphism manifested as occurrence of a cytosine residue atnucleotide residue 242 of the phox gene; xxii) a polymorphism manifestedas occurrence of a thymine residue at nucleotide residue 242 of the phoxgene; xxiii) a polymorphism manifested as occurrence of a 27 nucleotideresidue repeat between nucleotide residues 5130 and 5511 of the geneencoding nitric oxide synthase; xxiv) a polymorphism manifested asnon-occurrence of a 27 nucleotide residue repeat between nucleotideresidues 5130 and 5511 of the gene encoding nitric oxide synthase; xxv)a polymorphism manifested as occurrence of an adenine residue atnucleotide residue -290 of the gene encoding cytochrome P450 (i.e., thepolymorphism designated the CYP3A4 cytochrome P450 variant); xxvi) apolymorphism manifested as occurrence of a guanine residue at nucleotideresidue -290 of the gene encoding cytochrome P450 (i.e., thepolymorphism designated the CYP3A4 cytochrome P450 variant); xxvii) apolymorphism manifested as occurrence of a single guanine residue atnucleotide residue -1607 of the human gene encoding MMP-1; and xxviii) apolymorphism manifested as occurrence of a two consecutive guanineresidues at a site including nucleotide residue-1607 of the human geneencoding MMP-1, wherein the advisability that a human should consume orapply the product is determined based on occurrence of the assessedpolymorphisms.
 2. The method of claim 1, comprising assessing occurrencein the human's genome of disorder-associated polymorphisms in at leastfour of i) through xxviii).
 3. The method of claim 1, comprisingassessing occurrence in the human's genome of at least six of i) throughxxviii).
 4. The method of claim 1, comprising assessing occurrence inthe human's genome of at least ten of i) through xxviii).
 5. The methodof claim 1, comprising assessing occurrence in the human's genome of atleast fifteen of i) through xxviii).
 6. The method of claim 1,comprising assessing occurrence in the human's genome of each of i)through xxviii).
 7. The method of claim 1, wherein occurrence of anindividual disorder-associated polymorphism is assessed by contacting anucleic acid derived from the human's genome with a firstoligonucleotide that anneals with higher stringency with thedisorder-associated polymoiphism than with a correspondingnon-disorder-associated polymorphism and assessing annealing of thefirst oligonucleotide and the nucleic acid under hybridizationconditions sufficient to differentiate annealing of the firstoligonucleotide with the disorder-associated and non-disorder associatedpolymorphisms, whereby annealing of the first oligonucleotide and thenucleic acid is an indication that the human's genome comprises thedisorder-associated polymorphism.
 8. The method of claim 7, wherein thefirst oligonucleotide is attached to a support.
 9. The method of claim8, wherein the support has a plurality of different firstoligonucleotides attached thereto, wherein each oligonucleotide annealswith higher stringency with the disorder-associated polymorphism thanwith a corresponding non-disorder-associated polymorphism of a geneselected from the group.
 10. The method of claim 9, wherein the supporthas attached thereto at least five first oligonucleotides that annealwith higher stringency with the disorder-associated polymorphisms thanwith the corresponding non-disorder-associated polymorphisms.
 11. Themethod of claim 9, wherein the support has attached thereto at least tenfirst oligonucleotides that anneal with higher stringency with thedisorder-associated polymorphisms than with the correspondingnon-disorder-associated polymorphisms.
 12. The method of claim 9,wherein the support has attached thereto at least fifteen firstoligonucleotides that anneal with higher stringency with thedisorder-associated polymorphisms than with the correspondingnon-disorder-associated polymorphisms.
 13. The method of claim 7,wherein the first oligonucleotide is a molecular beacon oligonucleotide.14. The method of claim 7, wherein occurrence of an individualdisorder-associated polymorphism is further assessed by contacting thenucleic acid with a second oligonucleotide that anneals with higherstringency with a non-disorder-associated polymorphism than with thecorresponding non-disorder-associated polymorphism and assessingannealing of the second oligonucleotide and the nucleic acid underhybridization conditions sufficient to differentiate annealing of thefirst oligonucleotide with the disorder-associated and non-disorderassociated polymorphisms, whereby annealing of the secondoligonucleotide and the nucleic acid is an indication that the human'sgenome does not comprise the disorder-associated polymorphism.
 15. Themethod of claim 14, wherein the second oligonucleotide is attached to asupport.
 16. The method of claim 15, wherein the first and secondoligonucleotides are attached to the same support.
 17. The method ofclaim 14, wherein the second oligonucleotide is a molecular beaconoligonucleotide.
 18. The method of claim 17, wherein the first andsecond oligonucleotides are spectrally distinct molecular beaconoligonucleotides.